Abstract Background C hlamydia spp. are obligate, intracellular bacteria that infect humans and animals. Research on these important pathogens has been hindered due to a paucity of genetic tools. We recently adapted a group II intron (GII) mutagenesis platform for creation of ampicillin-selectable gene insertions in C. trachomatis L2. The aims of this study were: (1) to assess the stability of the intron-insertion in an in vivo infection model to gauge the efficacy of this genetic tool for long term animal studies and (2) to expand upon the utility of the method by validating a second selection marker ( aadA , conferring spectinomycin resistance) for mutant construction. Results Intron stability was assessed using a mouse vaginal tract infection model with a C. trachomatis L2 434/Bu incA ::GII( bla ) mutant. Infections were performed in the absence of selection and isolates shed into the vaginal tract were isolated and expanded in cell culture (also without selection). PCR and inclusion phenotype analysis indicated that the intron was stable for at least 27 days post-infection (at which point bacteria were no longer recovered from the mouse). The aminoglycoside 3′ adenyltransferase ( aadA ) gene was used to create a spectinomycin-selectable GII intron, facilitating the construction of an incA ::GII[ aadA ] C. trachomatis L2 insertion mutant. Both the GII( aadA ) intron and our previously reported GII( bla ) intron were then used to create an incA ::GII( aadA ), rsbV1 ::GII( bla ) double mutant. Mutants were confirmed via PCR, sequencing, inclusion morphology ( incA only), and western blot. Conclusions The stability of the intron-insertion during in vivo growth indicates that the GII-insertion mutants can be used to study pathogenesis using the well-established mouse infection model. In addition, the validation of an additional marker for mutagenesis in Chlamydia allows for gene complementation approaches and construction of targeted, double mutants in Chlamydia . The aadA marker also could be useful for other genetic methods. Collectively, our results expand upon the rapidly growing chlamydial genetic toolkit and will aid in the implementation of studies dissecting the contribution of individual genes to infection.